In Charcot-Marie-Tooth Syndrome (CMT) type 4H, a form of muscular dystrophy, peripheral nervous system glia (Schwann cells) form normally, but fail to maintain their structure, lose the ability to insulate neurons, and peripheral nervous system function is lost. The recently cloned human CMT4H protein is homologous to the C. elegans EXC-5 protein, the function of which is required to maintain the narrow tubules (excretory canals) of this genetic model nematode. Preliminary data suggest that EXC-5 regulates the passage of material from early endosomes to recycling endosomes, in order to rebuild constantly the luminal surface of this small tube;we believe the CMT4H is performing the same task in Schwann cells to maintain the tightly-wrapped tubule wrapped around the peripheral neurons. The goal of this project is to develop C. elegans as a genetic model for CMT4H, and to use the genetic tools available for C. elegans to identify proteins that function together with CMT4H to maintain tubule structure. Previous studies have identified one candidate interactor protein, and a trial genetic screen has already identified at least two additional such proteins. The screens are designed to identify suppressors of EXC-5 function. Interference with suppressor protein function should increase the rate of endosome recycling, with the potential to allow maintenance of Schwann cell morphology even in the absence of CMT4H function. The project will also provide insight to EXC-5/CMT4H function by examining the position of various endosomal markers within the nematode excretory canals in wild-type animals, in animals deficient in exc-5, in animals mutated for the interacting genes identified, and in animals where function of other CMT homologue proteins have been impaired via siRNA techniques. The model will also be tested by substituting human CMT4H for nematode EXC-5, to confirm that the functions of the proteins can substitute for one another. The C. elegans strains created through this work will be made available to the research community, and future grant applications will propose to use this new genetic model for high-throughput drug screens to identify compounds that can overcome loss of CMT4H function in nematodes, and potentially in CMT4H patients as well. PUBLIC HEALTH RELEVANCE: Charcot-Marie-Tooth Syndrome (CMT) is a series of diseases of the peripheral nerves that cause loss of sensation and muscular dystrophy. The CMT4H gene was cloned as FGD4, whose function is poorly understood in human cells. Genetic studies of the FGD4 homologue from the roundworm C. elegans suggest that the protein is necessary for recycling of endosomes in narrow single-celled tubules such as Schwann cells. This project will use C. elegans genetics to discover and investigate the proteins that interact with FGD4 to maintain peripheral nervous system function. Such a developed model has the potential to allow drug screening in living organisms to find compounds that can prevent onset of CMT symptoms.